Having reached the milestone of sequencing the human genome, the next question in understanding human biology is how genomes are organized in living cells and what the mechanisms are that determine what genome programs are active. The goal of our project is to analyze various aspects of gene expression in living cells using molecular tools in conjunction with microscopy techniques. A fundamental question in understanding how the human genome is organized and how genes are expressed is what the dynamic properties of nuclear structures and of nuclear proteins are in living cells. We are addressing these issues by generating functional, fluorescently labeled molecules, which can be introduced into cells and visualized in the nucleus of living cells by time-lapse microscopy. Using these methods we have succeeded in visualizing gene expression processes in living cells to study the structure-function relationship of the mammalian cell nucleus. Recently we have initiated studies to measure biophysical properties of proteins in vivo by using photobleaching techniques in conjunction with kinetic modeling and computer simulation approaches. These methods provide powerful tools to analyze for the first time at the molecular level the action of proteins in living cells and in real time The second major issue in understanding how genomes function in cells is to determining how genomes are organized. To this end we have initiated studies to map the spatial organization of chromosomes within the nucleus and we are testing whether positioning of chromosomes affects gene expression and regulation. These studies must be considered first steps towards rigorous interphase cytogenetic methods. Finally, we are using our cell biological approaches to investigate the cellular organization of alternative splicing by investigating the differential association of pre-mRNA splicing factors with alternatively spliced transcripts. We have also initiated a study to apply our knowledge of alternative splice site selection to correct aberrant splicing in human disease genes with the hope to restore normal splicing in a 'RNA-therapy' approach.